The invention relates in part to analytical instruments providing cost effective, automated testing for low to medium sample volume applications. The invention also relates in part to components, features, disposables, reagent delivery systems, accessories, and methods for using such instruments. The analytical instruments of the invention may be used for analytical testing, and in particular, for automated medical diagnostic testing. The invention describes a completely self-contained test surface and reagent delivery device that is used in conjunction with the instrument of the invention to perform an automated sample analysis. The instrument and cartridge system are well suited to the medical point of care testing environment or other analytical testing environments.
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Conventional automated clinical and chemical analyzers tend to be large, complex, multi-module instruments. For example, U.S. Pat. No. 5,902,548 describes an analyzer for high-throughput analysis. Such analyzers can be expensive, difficult to maintain, and require a significant amount of floor or bench space. Sample-processing is generally handled independent of the analyzer and requires manual placement of the processed sample into the sampling position of the analyzer. Such analyzers are not cost effective for the analysis of low sample volumes nor for providing single test results.
Most conventional analyzers use a modular approach to the various assay functions required to complete an assay procedure. For instance, one module may deliver a test device to a section of the analyzer for sample application. The next module would be used to introduce one or more reagents. Another module may be required to incubate the test device and another one may be required to wash the test device prior to the next cycle of reagent additions. A final module would be used to analyze the result generated within or on the test device. Some devices, such as that disclosed in U.S. Pat. No. 6,042,786 integrate a keyboard as an on-board instrument component. As discussed in U.S. Pat. No. 5,332,549, separate module may be required to remove the spent test device from the analyzer. Such designs require precise placement of the test device to insure proper operation within the analyzer. At the same time, the analyzer conveyance system must allow the test device to be placed and removed without binding within the carrier position. U.S. Pat. Nos. 5,167,922 and 5,219,526 describe an arrangement of test device and carrier features within an analyzer that serve to lock the test device into a carrier. With these types of analyzers the entire test device must be rotated or conveyed to different processing stations.
Many automated analyzers use some form of aspirator in combination with a probe or pipet tip device to automatically draw and dispense a sample or reagent from one container to a test container. For example, U.S. Pat. No. 5,983,734 discloses an analyzer with an aspiration-type sample delivery device. Similarly, U.S. Pat. No. 6,063,340 discloses an analyzer with aspiration and dispensing probes for sample and reagent delivery. To avoid contamination, the tips often must be disposed of after each reagent addition or washed prior to contact with the next solution. Disposing of tips after each use adds a high disposable cost to the instrument. Continuous washing of a reagent delivery system means there is generation of a high volume of liquid biohazardous/toxic waste that must be routinely disposed of. Multiple samplings of a single reagent container increases the possibility that the reagent will become contaminated. If the tip contacts the solution, the sides of the tip or probe may be covered with solution (sample or reagent). The residual solution may then be inappropriately dispensed to the test container or to another reagent container. The contamination of the next reagent may lead to improper assay results not only in the first test being conducted but in all subsequent tests using that reagent container. The use of an aspiration type device means that the reagent containers are exposed to the open environment leading to evaporation issues and potential contamination. Fluctuations in the aspiration system can lead to significant contamination of the entire reagent delivery mechanism and to variable fluid volumes being dispensed. Most systems use multiple sample reagent containers and dispense a unit of reagent with the initiation of each new assay and contain a separate module that contains the actual test device or surface.
Many automated analyzers use centrifugal force for the movement and volume control of reagents. Use of centrifugal force requires a radial array of reagents and precise fluid path constructions. Centrifugal force is used to drive fluid over a barrier and into the next reagent or reaction chamber until a detection member is encountered. High precision molding requirements make individual rotary test devices extremely expensive. Multiple fluid paths and reaction chambers within the fluid path to introduce new reagents and allow incubation time make the design of test devices even more difficult. Subjecting the test device to multiple bursts of centrifugal force can introduce errors in the flow of fluids along the desired pathway. U.S. Pat. No. 5,912,134 discloses an assay cartridge using channels, capillaries, reservoirs, and stop junctions to control reagent delivery, and sample dilution within the cartridge as a function of capillary, gravitational, and centrifugal forces.
A few assay systems have used discrete reagent containers, such as ampules or capsules or bags. The reagents are released by a breaking or piercing mechanism. Reagent delivery is then based on a passive gravity feed and thus can not ensure that the complete volume of the required reagent is dispensed. The breaking or piercing mechanism may also interfere with reagent delivery. If the breaking or piercing mechanism is in contact with more than one reagent container it is possible that it can carryover a reagent that affects the next reagent delivered to the test surface. In some cases, once the piercing member has penetrated the reagent container the fluid flows through a channel within the piercing member to be delivered to the test surface by capillary action or gravity feed. For example, U.S. Pat. No. 5,968,453 describes a reagent cartridge that is open to a sampling device for removal of reagent. Conversely, U.S. Pat. No. 6,043,097 describes a complex reagent container consisting of a sealed lid, and a valve that controls opening and closing of one or more chambers in the container. The reagent chamber holds a glass ampule that is crushed to release reagent, and a filter element.
Other cartridges use a pierceable member to exclude sample from the test cartridge until the member is pierced and then deliver a specific amount of sample by capillary action to the test cartridge. U.S. Pat. Nos. 5,888,826 or 5,602,037 describes a device where downward displacement of a vacuum chuck is used to press down on one section of the test cartridge. Lowering the sample cup of the test cartridge lowers a piercing member into the pierceable member. When vacuum is applied an amount of sample may be aspirated into the sample cup.
U.S. Pat. No. 4,689,204 describes a reagent delivery system that utilizes a series of plunger-like cylinders of varying heights for reagent delivery. As an upper plate-like actuator is depressed onto the various cylinders a sample or reagent is delivered to a reaction tube. The reagent delivery sequence is controlled by the height of the cylinders. The shorter the cylinder the later in the sequence the reagent is delivered. The reaction tube contains a coarse filter between sample addition to the reaction tube and the final reagent delivery to the reaction tube. At the end of the reaction tube is a fine filter to retain the analyte of interest, particularly bacteria. A lens is included in the reaction tube pathway for visualization of the fine filter.
In another embodiment of U.S. Pat. No. 4,689,204, the individual reagent chambers may consist of a piston-like member that when pushed into the reagent chamber drives the reagent past a pressure sensitive seal into a delivery tube. An actuating member pushes the piston-like member that also pierces the seal at the exit port to initiate fluid flow.
In both embodiments of U.S. Pat. No. 4,689,204, sample and reagent flow through the reaction tube is capillary or gravity flow and the actuation of each reagent is based on the linear progression of the actuating member as it passes each piston-like member. Each fluid has only the time between contact of the actuator with it""s specific piston-like member to the contact of the actuator with the next piston-like member to flow through the coarse filter and then the fine filter. The design of the coarse filter has a large open or dead volume or head space located above the coarse filter where premature mixing and interaction of the different fluids may occur. In addition this dead volume will retain a significant amount of fluid causing incomplete sample and reagent introduction to the fine filter.
U.S. Pat. No. 5,922,591 discloses an analytical device capable of collecting and analyzing a number of samples in a single unit. A pneumatic system is used to apply differential pressure for fluid movement.
Single use disposal diagnostic devices have been developed for a large number of applications, in particular for medical diagnostic applications. These tests provide timely single test results but require user intervention to produce the test results.
U.S. Pat. No. 5,006,309 describes a disposable device for use in an automated assay system. The device contains two wells. One well is used to process the sample and add reagents. The second well is used to read the assay result. The processed sample is transferred from one well to the other using jets of fluid. The processed sample consists of analyte and microparticles specifically reactive with the analyte. The processed sample is moved between wells without contacting a pipette or other transfer device. The sample well and the read well are connected by a fluid passage and processed sample is moved through the passage by a high velocity wash solution. The wash solution is introduced by a series of nozzles. The read well will retain a specific volume of the processed sample. The read well contains a fibrous matrix that will retain the processed sample. The flow of fluid through the fibrous matrix may be enhanced by the use of a vacuum or absorbent material under the matrix. The microparticle is used to specifically capture and retain the analyte. The fibrous matrix is selected to immobilize the microparticles within the fibrous matrix. Once the particles are immobilized a signal generating material is added to the matrix and the signal produced. The fibrous material must remain porous and support easy fluid flow once the microparticles are immobilized. Sample and reagents are added to the sample well through the use of pipettes and/or transfer devices that rely on aspiration mechanisms and are external to the disposable. Control of the wash solution speed is critical to effective functioning of the device.
Most assay devices or systems do not provide for on board processing of a sample collection device and even fewer systems can process more than one type of sample collection device or process more than one type of sample. U.S. Pat. No. 5,415,994 describes a manual assay device where the sample collection device is a swab. The specimen-containing swab is placed in a well within the assay device. Extraction reagents are added to the well containing the swab and allowed to flow past the swab extracting the analyte from the specimen on the swab. The solution continues to flow through the device to a test surface for analysis. The sample receiving position is joined to a bowl structure. The sample receiving position has a stop feature to properly position the swab above the bowl. The extraction chamber is in fluid contact with a sample receiving zone through an exit port. The matrix of the sample receiving zone defines the flow path from the extraction chamber. The extraction chamber is formed as an integral part of the solid device.
U.S. Pat. No. 5,084,245 describes a similar sample-processing device. The device is designed with a sample detection element in the base. The top of the device covers the sample detection element and contains an elongated feature positioned close to the sample detection element. This elongated feature is used to retain a swab carrying a sample. A number of extensions are contained within the elongated feature and used to squeeze or express fluid from the swab as it is pushed into the elongated feature. The expressed fluid then contacts the sample detection element. The top of the device must be removed to visualize the sample detection element. The extensions from the elongated passageway also serve to direct fluid flow to the surface of the sample detection element. In this invention the swab containing sample is not placed into the sample-processing device until it has been incubated in an extraction reagent and the extraction reagent has been allowed to saturate the fibers of the swab. The swab is inserted in the elongated feature until the tip is in fluid contact with the detection element.
U.S. Pat. No. 5,994,150 discloses an SPR-based detection system for optically analyzing a number of specific regions on a rotating platform.
Each of the foregoing U.S. Patents describing the background of the invention is hereby incorporated by reference in its entirety, including all tables, figures, and claims.
The present invention provides cost effective analytical instruments for determining the presence or amount of an analyte in a sample. The invention provides devices, instruments and methods useful to provide automated test results for single or low to medium sample volume applications, using an instrument that requires only a limited amount of laboratory space. The invention also addresses the technical limitations found in current automated analyzers by providing the analyzer with a test cartridge that contains all of the elements to conduct one or more assays or tests and provide results. The skilled artisan will readily appreciate that the test cartridge design of the invention is such that a number of different optical or electronic methods may be used to detect the analyte or provide a test result. The test cartridge of the invention is also designed to flexibly provide one or more analytical result.
The analytical, or medical point of care assay instrument and component aspects of the present invention provide self-contained sample processing and reagent capabilities for low to medium volume testing requirements. Representative testing applications include, but are not limited to, infectious disease testing, cancer detection and monitoring, genetic testing, therapeutic drug level monitoring, allergy testing, environmental testing, food testing, diagnostic and/or prognostic testing of human and veterinary samples, off-line process testing, etc. Preferably, the instrument uses an optical detection method based on a fixed polarizer ellipsometric method and a test surface designed for analysis of thin films. Particularly preferred embodiments of such methods and test surfaces are described in U.S. Pat. Nos. 5,494,829 and 5,631,171, which are hereby incorporated by reference in their entirety, including all figures. In certain of these embodiments, the test devices use a combination of thin films to modify the reflection of light from the surface of the test device. Alternatively, the instruments use a detection system, for example a spectrophotometric, chemilluminscent, fluorescent, or electrical potential detection method, that is consistent with the test surface, supporting reagents contained within an assay cartridge, and the signal produced from the test surface.
Assay cartridges are preferably single-use disposable elements designed to conduct a specific type of analysis. Such cartridges contain features that allow for multiple reagents to be stored separately within the cartridge unit. The cartridge also typically contains features that, in conjunction with instrument elements, can result in the delivery of those reagents in the proper sequence to the test surface. In these embodiments, the cartridge is capable of receiving a sample and, in conjunction with instrument elements, processing the sample for application to the test surface. A sample processing element of the cartridge includes the ability to receive and retain a sample collection device, e.g., a swab or a sample reservoir. In preferred embodiments, the sample processing element can retain the sample collection device in a stable configuration as the cartridge, or an element thereof, is indexed or moved to various analysis positions.
The sample processing element can be a separate component that is inserted into the cartridge during manufacturing, or attached by the user immediately prior to use. Alternatively, the sample processing element can be an integral part of the cartridge. Thus, while only a single cartridge design is required, the sample-processing element can be uniquely tailored to accept a wide range of sample collection devices or sample types. The selection of the sample processing element to be inserted into, or otherwise associated with, the cartridge is a function of the specific analysis the cartridge is designed to conduct. The sample processing element can be designed to accommodate a specific sample type, or may be designed to accommodate multiple sample types. In preferred embodiments, the sample processing element comprises a hinged extension designed to support the shaft of a swab-type sample collection device. When used, the hinged extension can prevent the swab shaft from being inadvertently dislodged, and can act as a signal to the instrument that a swab is in use. In other preferred embodiments, a filtration membrane is positioned below a small opening in the sample processing element through which a sample fluid must flow.
Assay cartridges of the invention are also designed to deliver the sample and the assay reagents to a test surface within the cartridge. The assay cartridge, in conjunction with the instrument, may be indexed in a specific sequence to allow the proper addition of reagents to the test surface or other regions of the cartridge. The instrument preferably indexes the cartridge such that the test surface is available for analysis at one or more stages in the assay process. The detection system included in the instrument is designed to be compatible with the type of test surface and reagents contained in the cartridge. For example, when the test surface is a membrane coated with an analyte-specific binding reagent and one of the reagents is an anti-analyte antibody derivatized with a fluorescent label, then the instrument can contain a fluorimeter for analysis. If the test surface is a series of micro-electrodes and the reagents are redox type reagents, the instrument can contain a detection system which provides a potentiometric result. In preferred embodiments, the assay cartridge is capable of generating a signal without addition of external signal-related reagents. For ease of presentation only, the test surface primarily discussed is an optically active test surface. However, those skilled in the art will recognize the flexibility and capabilities of test cartridge design and how to match those properties to a specific detection method within an automated instrument system of the invention.
Thus, in preferred embodiments this invention concerns assay cartridges that preferably comprise a bottom member and a top member, the bottom member comprising an optical reading well and a test surface, and the top member comprising a rotatable reagent carousel. The reagent carousel comprises a sample receiving port and a plurality of reagent wells. The reagent carousel has an opening that is aligned with the optical reading well containing the test surface when an analysis of the surface is to be performed. One or more reagent wells comprise a reagent and a reagent well piston for delivery of one or more reagents to the test surface and/or the sample receiving port. The top member attaches to the bottom member such that the reagent carousel may be rotated relative to the bottom member.
In another aspect, the invention concerns methods for producing an assay cartridge for a specific analysis and sample type. The assay cartridge can be fabricated using manufacturing techniques which are well known in the art. Preferably, the assay cartridge is fabricated by attaching the bottom member to the top member such that the rotatable reagent carousel may be rotated relative to the bottom member. Particularly preferred methods of fabricating the assay cartridges of the invention are described herein.
Yet another aspect of the invention concerns test kits for use with an analytical assay instrument, the test kit preferably comprising a number of assay cartridges specific to the analyte(s) the kit is designed to detect, and instructions for their use. Preferably, the kit includes one or more external kit control elements for additional quality control of the test procedure and equipment. Most preferably, such kits include appropriate sample collection device(s) (e.g., swabs, liquid sampling cups, etc.) which are consistent with the sample requirements for the types of analytes to be detected using the assay cartridges of the kit.
In particularly preferred embodiments, the assay cartridge comprises one or more of the following: (i) a test surface comprising an analyte-specific binding layer for immobilizing an analyte on the test surface; (ii) a test surface that nonspecifically immobilizes an analyte thereon; (iii) a test surface that is an optically active test surface; (iv) an optically active test surface that is adapted to generate an interference, ellipsometric, or polarization signal; (v) a rotatable carousel that further comprises a sample processing element; (vi) a sample processing element comprising a filtration surface; (vii) a sample receiving port adapted to receive a swab type sample collection device; (viii) a bottom portion of each reagent well sealed by a breakable seal material, and a top portion of each reagent well sealed by the reagent well piston in combination with a breakable seal material; (ix) reagent well piston(s) comprising a piercing element to break the breakable seal material sealing the bottom of the reagent well; (x) a bottom member comprising extender tabs or other mechanism adapted to ensure proper registration of the assay cartridge with an analytical instrument; and (xi) reagent well piston(s) comprising a hex boss element.
The assay cartridge is preferably made from a plastic material, such as polystyrene, which provides mechanical strength and stability. The skilled artisan will recognize that such assay cartridges, or components thereof, may be made from a number of thermoplastics which are suitable for injection molding. In preferred embodiments, the bottom member of the assay cartridge is made from a single piece of a plastic material. Most preferably, the bottom member of the assay cartridge comprises an upper and a lower section which are mated together. The top element comprising a reagent carousel can be made from any suitable material, preferably polyethylene that is stiffened with talc, to facilitate handling characteristics during manufacture. The reagent carousel is attached to the top member of the assay cartridge in a manner which allows for rotation of the reagent carousel relative to the bottom member.
The term xe2x80x9csamplexe2x80x9d as used herein refers to any specimen suitable for analysis within an assay cartridge according to the invention. Preferred sample types include, but are not limited to, a material, including biological material, collected on swabs (e.g., throat, vaginal, endocervical, rectal, urethral, nasal, or nasopharyngeal swabs), fluids, water, urine, blood, sputum, serum, plasma, fecal material, aspirates, washes, tissue homogenates or samples, process fluids, etc.
The term xe2x80x9csample collection devicexe2x80x9d as used herein refers to any support used for transfer of a sample into the device. Suitable sample collection devices are well known to those skilled in the art. Preferably, a sample collection device can be a swab, a wooden spatula, bibulous materials such as a cotton ball, filter, or gauze pad, an absorbent-tipped applicator, capillery tube, and a pipet.
A vacuum element can be used to express the sample from the sample collection device and to deliver the sample, or a portion thereof, to the optically active test surface. In preferred embodiments, the same vacuum source can be used to secure the cartridge to the instrument""s cartridge platform and/or to promote sample and reagent flow through or over or around the optically active test surface. Alternatively, cartridge locking and positioning can also be accomplished by a mechanical element, such as locking mechanisms, or set pins. One or more different vacuum elements may also be used in order to separate the various vacuum functions that may be required to complete a particular assay.
A sample may be processed prior to moving from the sample processing element of the cartridge to the optically active test surface. To process the sample, one or more reagents are added to the sample collection device within the sample processing element. These reagents serve to extract, or free, analyte from the sample collection device and from the sample matrix or from an organism contained on the sample collection device. The reagents may assist in eliminating sample matrix effects such as inhibition or non-specific binding. The sample-processing element may also include a filter feature to remove particulates from a sample prior to introduction to the test surface.
In other preferred embodiments, the sample is processed, for example by filtration with or without subsequent extraction, prior to introducing the sample to the optically active surface. When a fluid such as urine or a suspension contains the analyte, the analyte may be retained on a sample processing element that contains a filtration surface. The sample is added to the sample receiving/processing assembly when the reagent carousel is rotated above an absorbent material located in the base of the cartridge. The absorbent material serves as a self-contained waste reservoir and does not expose other cartridge elements to the waste material. The sample fluid is drawn through the filtration surface by application of vacuum or other pressure differential. Once the sample fluid is filtered, extraction reagents can be applied to the filtration surface. The analyte is then solubilized within the extraction reagent prior to introduction to the optically active test surface. The sample-processing element is indexed to the test surface position and sample delivered. Assay processing proceeds as described herein.
The term xe2x80x9canalytexe2x80x9d as used herein refers to any material that is a specific indicator of a disease, infection, drug level, analytical condition, environmental condition, process condition, medical condition, or any other condition that can be diagnosed or assessed by rapid, sensitive detection of the presence or amount of the analyte. Preferably, an analyte is an antigen, antibody, nucleic acid, metal, receptor, enzyme, enzyme substrate, enzyme inhibitor, ligand, chelator, hapten, drug, or analog, or any fragment of these materials.
In further particularly preferred embodiments, the assay cartridge comprises: (i) a sample receiving port capable of receiving a volume of fluid sample onto a concentrating element that is unique to the sample collection device or sample type; (ii) a sample receiving port comprising a retaining mechanism (e.g., molded fingers) to hold a swab type sample collection device in the proper position in the reagent carousel for subsequent processing; (iii) an optically active test surface positioned in the base of the cartridge, consisting of a low porosity material having one or more optical layers positioned thereon to create a surface with the proper optical characteristics for the detection method built into the instrument; and (iv) a reagent carousel in a plastic housing comprising one or more reagents.
The term xe2x80x9csample receiving portxe2x80x9d as used herein refers to an opening in the assay cartridge which provides access to the interior of the cartridge. A suitable sample receiving port can be readily determined by one skilled in the art, based on the type of sample and/or sample collection device.
As discussed above, the sample processing element can be a separate component that is inserted into the cartridge during manufacturing or attached by the user, or can be an integral part of the cartridge. The sample processing element is preferably designed to accommodate a specific sample type for a specific test method and analyte. In preferred embodiments, a sample delivery port is fully integrated into a reagent carousel section of the cartridge, and contains an appropriate reagent delivery configuration. In other preferred embodiments, the reagent delivery configuration allows an extraction reagent to flow into a groove or channel at the top of the sample receiving port for extraction of an analyte from the sample collection element.
Preferably, a swab is used as a sample collection device, and the sample processing element comprises a swab holder or a swab processing insert. The swab holder or swab processing insert can be tapered or angled to allow a single sample processing element to accommodate all types of swabs by allowing swabs with different amounts of fiber, or that are wound to different levels of tightness, to be held securely within the holder or insert. Most preferably, the swab holder or swab processing insert securely holds the swab to provide stability during reagent cartridge indexing, and to provide a vacuum seal to assist in fluid flow around and through the swab.
The term xe2x80x9ctest surfacexe2x80x9d as used herein refers to a surface within the assay cartridge which is adapted to provide a detectable signal corresponding to the presence or amount of an analyte in a sample. Most preferred are optically active test surfaces, as defined herein. The test surface can be made available to the detector element of the instrument through an optical reading well in the upper section of the assay cartridge. The term xe2x80x9coptical readingxe2x80x9d well as used herein refers to an aperture or opening in the assay cartridge through which the test surface can be optically read or analyzed by a detector appropriate for the type of signal generated at or by the test surface.
When designing and constructing a test surface according to the invention, it is preferred that such a surface be adapted to specifically bind an analyte of interest, unless an analyte which is nonspecifically immobilized on the test surface can be specifically detected. Therefore, the test surface preferably comprises an analyte-specific binding layer to immobilize one or more analytes of interest on the test surface. The analyte-specific binding layer may be any material that will specifically interact with an analyte in a test matrix and retain that analyte throughout the assay procedure, or until a signal is detected from the test surface. Most preferably, an analyte-specific binding layer can comprise an antibody, antigen, a nucleic acid, enzyme, enzyme substrate, enzyme inhibitor, receptor, ligand, metal, chelator, complexing agent, hapten, or analog, or a fragment of any of these materials. In construction of an optically active test surface, it may be advantageous to add a layer of material to provide long term stability of the analyte-specific binding reagent. This layer is removed during the assay procedure or does not interfere with the assay procedure.
The analyte-specific binding layer may be applied to a test surface, preferably an optically active surface, by a number of different processes. The skilled artisan will recognize that such processes will depend on the nature of the molecules to be employed to specifically bind the analyte(s). In preferred embodiments, the analyte-specific binding layer is coated to the entire surface by submersion in a liquid coating solution, or applied by micro-spotting, ink jetting, or other printing type processes. The analyte-specific binding layer may be applied as a single spot of a specific diameter determined by the volume and viscosity of the coating solution and the wettability of the surface. The analyte-specific binding layer may be applied as a line or other symbol using commercially available processing equipment.
In other particularly preferred embodiments, test surfaces of the invention comprise a plurality of analyte-specific binding layers, each comprising one or more binding reagents specific for a different analyte. Preferably, binding reagents are applied to the test surface in a plurality of zones, thus allowing for the detection of multiple analytes from a single sample in a single analysis. Thus, the analyte-specific binding layer can preferably be applied as a series of stripes, dots, or other symbols in any desired array. The size of the array placed on the test surface is limited by the available test surface area, the spatial resolution required to uniquely identify each position within the array, the detectors spatial resolution capabilities, and the spatial resolution of applying the analyte-specific binding layers. In addition to the analyte-specific binding layer, various analysis controls, e.g., positive and/or negative control zones, can also be applied to the test surface for use in quality control of the test result.
To improve the sensitivity of the testing method, once an analyte is associated with its analyte-specific binding reagent on the test surface, a secondary reagent that includes an analyte-specific binding reagent may be used. This additional analyte-specific reagent may include additional reagents specifically associated with it to amplify the binding of analyte to the optically active test surface or when other surface constructions are used to provide for signal generation. Preferably, these additional reagents are selected from the group consisting of enzymes, film forming particles, catalytic reagents producing an insoluble product, self-assembling molecules, or other materials that will increase the optical thickness of the analyte layer. When the test surface construction does not include optically functional layers, the amplifying reagents are solely responsible for signal generation. If an analyte-specific binding reagent is not used on the test surface, the analyte may be retained by nonspecific interaction with the test surface. Specificity for this type of assay is obtained with a secondary analyte-specific reagent.
The term xe2x80x9coptically active test surfacexe2x80x9d as used herein refers to a test surface which is adapted to alter incident light. Incident light refers to any electromagentic radiation which impinges on the surface. Preferably, incident light is unpolarized light, polarized light, eliptically polarized light, linearly polarized light, monochromatic light, polychromatic light, visible light, ultraviolet light, and infrared light. Methods for preparing an optically active test surface are known to those skilled in the art. Preferred methods for preparing an optically active test surface are described in PCT International Publication Number WO 94/03774 and U.S. patent application Ser. No. 08/950,963, filed Oct. 15, 1997 each of which is hereby incorporated by reference in its entirety, including all figures, or according to similar optical principles. Preferably, the optically active surface is sealed into a position in the base of the cartridge such it is not distorted by the application of the vacuum source. Particularly preferred sealing processes are heat sealing, pressure sensitive adhesives, adhesives, sonic welding, or ultrasonics, and similar processes.
The term xe2x80x9coptically functional layerxe2x80x9d as used herein refers to a layer (or layers) that can produce a signal upon binding of an analyte to the receptive material. The layer may have one or more coatings, including a base layer with or without an antireflective (AR) layer, designed to modify the optical properties of the support material so that the desired degree of reflectivity, transmittance, and/or absorbance suited to the final assay configuration is obtained. The optically functional layer may attenuate one or more, or a range of wavelengths of light so that a result is observable in an instrumented analysis in the final device upon analyte binding. The attenuation of light may involve the extinction or enhancement of specific wavelengths of light as in an AR coated assay device for a visually observable color change. Or the intensity of a specific wavelength of light may be modified upon reflection or transmittance from the final assay device. The generation of an AR effect is not required for the instrumented detection of the thin film effect. In all cases the optically functional layer serves to attenuate the light incident on the optically active test surface through the interaction of the light with the thin films on the optically active test surface. The optically functional layer may also modify the optical parameters of the device to allow a change in the state or degree of polarization in the incident light. Optically functional layers include amorphous silicon, silicon nitride, diamond like carbon, titanium, titanium dioxide, silicon dioxide, silicon carbide, silicon oxynitride, silicon monoxide, and other related materials or composites of these materials. A preferred construction of optically functional layers is a layer of amorphous silicon coated onto a polycarbonate membrane and then coated with a layer of diamond like carbon. Another preferred construction of the optically functional layers is a layer of amorphous silicon coated onto a polycarbonate membrane then coated with a layer of silicon nitride and a thin layer of diamond-like carbon. Optically functional materials may be applied to the support material by sputtering, ion beam deposition, vapor deposition, spin coating, direct current plasma, chemical vapor deposition, or other methods known to those skilled in the art.
The base optical layer serves to provide the optical characteristics required for creating the appropriate reflectance, adsorption, or transmission properties. It must be sufficiently dense to eliminate stray light leakage or back scattering from the backside of the support. As the thickness of the base layer increases so will the percent reflectance of the modified support. The desired percent reflectivity will depend on the optical system incorporated into the instrument. Appropriate base layer material includes amorphous silicon, polycrystalline silicon, lead telluride, titanium, germanium, chromium, cobalt, gallium, tellurium, or iron oxide. The final optical properties of the optically active test surface are optimized to consider the optical contribution of all layers of the final test surface. Thus, the base optical layer may be adjusted based on empirical testing or thin film reflection theory modeling to account for the attachment layer or the analyte-specific binding layer or any other layer that will be present in the final optically active test surface.
The optically functional layer may serve to provide the desired optical properties and may also serve as an attachment layer. An additional layer may be applied in the construction of the optically active test surface that serves the sole purpose of an attachment layer.
In particularly preferred embodiments, the optically active surface: (i) has an analyte-specific binding reagent immobilized on the surface; (ii) is able to non-specifically capture the analyte to be detected; (iii) is reflective and capable of generating an interference signal upon addition of a specific analyte or target to the optically active surface during performance of the assay steps; (iv) is reflective and capable of generating an ellipsometric signal upon addition of a specific analyte or target to the optically active surface during performance of the assay steps; (v) is reflective and capable of generating a polarization signal upon addition of a specific analyte or target to the optically active surface during performance of the assay steps; and (vi) the interference, ellipsometric, or polarization signal is related to the presence or amount of the specific analyte or target.
An additional attachment layer may be applied to the optical materials to improve their binding and retention of the analyte-specific binding layer or to other types of test surfaces as well. An attachment layer is any material or combination of materials that promote or increase the binding of the receptive material to the optically functional layer. Also, the attachment layer should retain the receptive material with sufficient avidity for all subsequent processing and assay steps. Preferably, the attachment layer should not reduce the stability of the receptive material and should insulate the receptive material from the optically functional layer or layers thereby improving the stability of the receptive material. When no receptive layer is utilized, the attachment layer may be used to non-specifically bind the analyte of interest. Attachment layers can be constructed of silanes, siloxanes, polymeric materials, nickel, diamond-like carbon, and the like. The attachment layer may be applied by vapor phase deposition, solution coating, spin coating, spray coating, a printing-type process, or other methods known in the art. A list of appropriate attachment layers and ways to identify attachments layers is described in U.S. Pat. No. 5,468,606 incorporated herein by reference in its entirety.
The attachment layer should also assist in the stabilization of the analyte-specific binding layer. When an attachment layer is employed, the material can be applied by exposure of the test surface to a vapor of the material under vacuum. Or the layer may be created by solution coating, by spin coating, by ink jetting, by printing processes, or other methods for application of a thin layer of the desired material. Once the material is applied to the test surface, a curing step may be employed to ensure permanent adhesion of the layer to the test surface. Curing is generally accomplished by exposure of the test surface to an elevated temperature for a period of time. The thickness of the attachment layer preferably provides sufficient density to the analyte-specific binding layer and separates the binding layer from the test surface, particularly when an optically active test surface is used. The attachment layer is then applied to the optical materials. The attachment layer may be used in some applications for the nonspecific capture of the analyte of interest. Construction of test surfaces other than optically active test surfaces may not require the use of an attachment layer.
The terms xe2x80x9cfilmxe2x80x9d and xe2x80x9cthin filmxe2x80x9d as used herein refer to a one or more layers of sample material deposited on a substrate surface. A film can be about 1 xc3x85 in thickness, about 5 xc3x85 in thickness, about 10 xc3x85 in thickness, about 25 xc3x85 in thickness, about 50 xc3x85 in thickness, about 100 xc3x85 in thickness, about 200 xc3x85 in thickness, about 350 xc3x85 in thickness, about 500 xc3x85 in thickness, about 750 xc3x85 in thickness, about 1000 xc3x85 in thickness, and about 2000 xc3x85 in thickness. Particularly preferred are films from about 5 xc3x85 to about 1000 xc3x85 in thickness; most preferred are films from about 5 xc3x85 to about 750 xc3x85 in thickness.
In other particularly preferred embodiments, the reagent carousel: (i) can be freely rotated on the bottom member through about 90xc2x0, 120xc2x0, 150xc2x0, 180xc2x0, 210xc2x0, 240xc2x0, 270xc2x0, 300xc2x0, 330xc2x0, and most preferably 360xc2x0, without binding or catching on any other portion of the instrument when the sample collection device is inserted in the sample receiving port; (ii) is mated to the upper surface of the bottom section of the assay cartridge, where the bottom section is formed of two molded plastic articles that may be sealed to create the bottom section of the overall assay cartridge; (iii) the upper surface of the bottom section includes one or more elements such as extender tabs or set pins; and (iv) the bottom surface of the bottom section contains one or more elements such as indentations for ensuring that the cartridge is in the proper position and orientation to conduct the assay method as well as improve the users"" grip on the test cartridge when loading on the instrument.
The term xe2x80x9cextender tabsxe2x80x9d as used herein refers to elements which extend from the assay cartridge and assist in orienting the cartridge within the instrument. Extender tabs are in the raised position in the final assembled assay cartridge and serve to lock the reagent carousel into place. When the cartridge is in proper registration within the instrument, the tabs are pushed down and the reagent carousel is free to rotate. The cartridge may be held in place in the instrument by a variety of mechanisms well known in the art. Preferred means of providing alignment and stability of the cartridge can be application of vacuum, by means of force applied by a presser foot, release arms and/or by a lock and key type matching of the cartridge bottom to the instrument cartridge slot, or simple matching of set pins of sufficient height to stabilize and retain the cartridge.
In other particularly preferred embodiments: (i) reagents are sealed within the reagent carousel by a thin layer of a breakable vapor seal material at the bottom of a reagent well; (ii) reagents are sealed within the reagent carousel by a thin layer of impermeable, vapor seal material at the upper opening of the reagent well; (iii) the upper reagent well seal is in contact with a reagent well piston; (iv) the reagent well piston is a rigid (e.g., plastic) element designed with a receiving element for a plunger element for driving the piston (e.g., a hex boss) in the uppermost segment of the piston, such that the uppermost segment of the piston extends above the upper surface of the reagent carousel section of the cartridge; (v) the hex boss is designed to mate with a push rod of the plunger element in the instrument housing; (vi) the push rod mates with the hex boss, or other receiving element of the piston, and a vertical drive element pushes the piston through the lower impermeable vapor seal to release reagent onto the optically active surface; (vi) the plunger element draws the piston back into the upper position to allow proper motion of the cartridge to the next assay position; and (viii) the plunger element includes an optional presser foot to improve registration of the reagent carousel and the membrane holder within the cartridge.
In another particularly preferred embodiment, the reagent well piston is pushed down with a plunger element, but is not equipped with a receiving element for said plunger. Instead, the plunger pushes the piston by contacting the uppermost surface of the piston without mating to a element such as a hex boss. As the plunger does not mate with the piston, it does not draw the piston back into the upper position, but allows it to stay in the depressed position.
The term xe2x80x9cvapor seal materialxe2x80x9d as used herein refers to a breakable sealing material which provides a liquid- and vapor-impermeable barrier at the top and/or bottom of each reagent well. The vapor seal material is intended to be broken by application of force by a reagent well piston at an appropriate point in the assay procedure, in order to provide flow of reagent. Suitable vapor seal materials are well known to those skilled in the art. Particularly preferred vapor seal materials are mylar and low density polyethylene. In preferred embodiments, the vapor seal material is affixed to the reagent well by an adhesive. The vapor seal material may also comprise additional layers of material such as foils, papers, additional plastics, and the like. Preferably, the vapor seal material comprises a layer of 15 pound polyethylene, a layer of aluminum foil, a layer of 7.2 pound polyethylene, and a layer of 25 pound ClF coated paper (Genesis Converting Corporation). Those skilled in the art will recognize that other materials of similar composition may substitute in the vapor seal material.
The term xe2x80x9creagent wellxe2x80x9d as used herein refers to a chamber in the carousel that contains a reagent for use in an assay procedure. A reagent may be any suitable reagent, including but not limited to a wash reagent, a buffer reagent, an extraction reagent, a neutralizing reagent, an amplifying reagent, or a signal generating reagent, as defined herein.
The term xe2x80x9creagent well pistonxe2x80x9d as used herein refers to an element that provides positive pressure to the reagent well for delivery of a reagent. A preferred material for the reagent piston is polycarbonate. The reagent well piston is pushed by a plunger mechanism of the instrument, creating sufficient force to break the lower seal of the reagent well and deliver fluid from the reagent well. The reagent well piston may contain an element to ensure positive engagement by the plunger mechanism. In a preferred embodiment, the element that ensures positive engagement is a hex boss. The reagent well piston may comprise a piercing element to assist in breaking the lower reagent well seal. The reagent well piston may or may not need to be retracted back into the reagent well once it is used to pierce the reagent seal, depending on the carousel design and whether the piston will prevent reagent carousel rotation. A piston design that need not be retracted may not require the hex boss element, as it need not seat with the push rod mechanism. The reagent well piston can also serve to assist in sealing the upper portion of the reagent well.
The piston design, the rate that the plunger mechanism displaces the piston in the reagent well, and/or the aperture size for the reagent well can allow for control of the reagent flow to the test surface. Piston design and displacement can also be used to control the amount of reagent delivered to the test surface. The piston may be designed with grooves of various shapes and sizes and numbers. The contour and number of grooves in the piston will modify the fluid flow rate through interactions such as surface tension and retention of the fluid contact with piston material as the piston is displaced. The piston design and the rate of displacement, as well as the materials in the lower vapor seal, determine the aperture size for the dispensing of the reagents. The quality of the aperture is also important in determining fluid flow rate. The quality of the aperture generated means the size of the opening, the structure of the opening, the cleanliness of the opening, etc. The piston design and the lower vapor seal as well as the displacement rate of the piston must be evaluated together to optimization reagent dispensing.
The term xe2x80x9chex bossxe2x80x9d as used herein refers to a raised element at the top of the reagent well piston comprising a hexagonally-shaped recess. In preferred embodiments, the plunger mechanism mates with the hexagonal recess to ensure positive engagement of the reagent well piston by the plunger. Those skilled in the art will recognize that the recess need not be hexagonally shaped, but rather can be any shape which is capable of mating with the plunger mechanism. The plunger mechanism can also be designed with spring-loaded mechanisms to control the push rod. Release of tension on the spring mechanism allows the push rod to displace the piston. If different pressures are required to break the reagent seals concentric push rods can be designed with different spring tensions to deliver varying displacement capabilities.
In another aspect, the invention concerns analytical instruments that comprise or utilize assay cartridges according to the invention, a mechanism, element or subassembly for receiving the assay cartridge, one or more rotation elements or subassembly to rotate and index the reagent carousel, a plunger element or subassembly for engaging the reagent well pistons to deliver reagent from the reagent wells to the sample receiving port and/or the test surface, a vacuum element or subassembly for directing sample and/or reagent to the test surface, and a detector for detecting a signal from the test surface. Preferably, a control processor controls the rotating, plunger, and vacuum elements according to an assay algorithm, and a signal processor for relating the generated signal to the presence or amount of an analyte.
In particularly preferred embodiments, the analytical instruments of the invention comprise one or more of the following: (i) a presser foot for stabilizing the assay cartridge; (ii) an optical control element for determining cartridge orientation; (iii) a rotation element comprising a mechanical arm and motor; (iv) a plunger element comprising a push rod attached to a vertical drive element; (v) a push rod adapted to seat in a hex boss element on the reagent well piston; (vi) a push rod that returns the reagent well piston to about its original position in the reagent well following delivery of the reagent; (vii) a detector selected from the group consisting of a color sensor, a color detector, an image detector, a spectrophotometer, a luminometer, a fluorometer, a potentiometer, an interferometer, a polarimeter, and an ellipsometet; (viii) a detector that is a fixed polarizer ellipsometer; (ix) a control processor and a signal processor consisting of a single general purpose computer programmed to perform instrument control and data processing algorithms; (x) an assay cartridge comprising an identifying element which identifies the analyte and/or the sample to the analytical instrument; (xi) an assay cartridge comprising an identifying element that is a bar code, and a bar code reader configured to read the bar code; and (xii) a sample receiving port adapted to receive a swab type sample collection device.
The analytic instruments preferably comprise an element for detecting a signal from the test surface. Depending upon the type of assay to be performed, the detector can be a color sensor, a color detector, an image detector, a spectrophotometer, a luminometer, a fluorometer, a potentiometer, an interferometer, a polarimeter, and an ellipsometer. One skilled in the art can readily match a suitable detection element to the assay being performed. Most preferably, the detection element is a fixed angle ellipsometer.
The term xe2x80x9cinterference signalxe2x80x9d as used herein refers to a change in the wavelength (xe2x80x9ccolorxe2x80x9d) of light reflected by an optically active surface, due to changes in the optical thickness of the sample material adsorbed or specifically bound to the surface. Interference may be measured and related to the presence or amount of the specific analyte or target by techniques that are well known in the art.
The term xe2x80x9cellipsometric signalxe2x80x9d as used herein refers to a change in the elliptical polarization of light reflected by an optically active surface, due to changes in optical thickness of the sample material adsorbed or specifically bound to the surface. An ellips6metric signal may be measured and related to the presence or amount of the specific analyte or target by techniques that are well known in the art.
The term xe2x80x9cpolarization signalxe2x80x9d as used herein refers to a change in the linear polarization of light reflected by an optically active surface, due to changes in optical thickness of the sample material adsorbed or specifically bound to the surface. A polarization signal may be measured and related to the presence or amount of the specific analyte or target by techniques that are well known in the art.
In particularly preferred embodiments, the analytical instrument: (i) comprises a user interface element; (ii) comprises a control processor; (iii) comprises a signal processor; (iv) comprises an algorithm for signal processing and data classification; (v) comprises an algorithm for determining an assay sequence; (vi) receives one or more assay cartridge(s) and completes the assay protocol independent of the user; (vii) mechanically indexes the assay cartridge so that the optically active surface of the cartridge is available for analysis at one or more stages in the analysis process; (viii) comprises a carousel rotation element consisting of a mechanical arm and a motor which indexes the reagent carousel to different positions for delivery of reagents to the optically active surface of the cartridge in the appropriate sequence; and (ix) comprises one or more optical control elements, such as optical encoders or bar code readers, for reagent carousel indexing, cartridge positioning, and determining cartridge orientation, and establishing the analytical method to be used and type of result to be reported.
The term xe2x80x9cuser interfacexe2x80x9d as used herein refers to an element of the instrument which allows the user to provide information and/or instructions to the device, and/or for the device to provide information and/or instructions to the operator. Those skilled in the art will recognize appropriate user interfaces. For example, a user interface can be one or more of the following: a bar code reader, a keyboard, a computer xe2x80x9cmouse,xe2x80x9d a light pen, a computer screen, and a computer printer.
The term xe2x80x9calgorithmxe2x80x9d as used herein refers to a sequence of steps to be followed to perform an assay and/or analyze data obtained from an assay. In preferred embodiments, an algorithm is stored on a control processor which controls the operation of the analytical instrument, and/or a signal processor which processes a signal generated from the test surface into a meaningful assay result. Preferably, the control and signal processors are one or more general purpose computer elements or computer chips which are programmed with the appropriate algorithm.
The term xe2x80x9cdaemonxe2x80x9d as used herein refers to a process that occurs in the background and is invisible to the user. Preferably, daemons run continuously throughout the assay procedure. A daemon may also be referred to as a background procedure or a background thread of execution.
The term xe2x80x9cindexxe2x80x9d as used herein refers to positioning of an assay cartridge in specific orientations. A cartridge can be indexed so that discrete locations on the cartridge, for example a reagent well and the test surface, precisely align with one another for properly timed and/or positioned reagent delivery. Preferably, analytical instruments of the invention use a mechanical mechanism or subassembly for cartridge indexing.
The term xe2x80x9coptical control elementsxe2x80x9d as used herein refers to a optical sensor mechanism which determines the orientation of a cartridge element. Appropriate optical control elements are well known in the art.
Preferably, one or more parameters required for proper sample processing can be provided by the user through the user interface of the instrument. For example, a user may indicate the sample type, the type of sample collection device, and/or the assay protocol. Most preferably, however, the assay cartridge is configured during manufacture such that each combination of assay type, sample collection device, etc., is represented by a distinct assay cartridge which can be recognized by the instrument and distinguished from other cartridges. The distinct assay cartridge can provide the appropriate reagent carousel for the assay, as well as the sample retention and extraction mechanism required by a given sample collection device. For example, for swab-type sample collection devices, a sample retention mechanism must also serve to direct extraction fluid into the swab fibers and not just around the swab fiber. Assay sequence, incubation times, and other parameters can be pre-set for each cartridge design. Cartridge lot information may also prompt the instrument to select the proper assay parameters and sequence as well as data analysis method. Alternatively, data processing may occur manually by the user.
In other particularly preferred embodiments, the vacuum element of the analytical instrument: (i) comprises a single vacuum source having one or more vacuum ports; (ii) uses vacuum for cartridge retention and stability, reagent flow through, sample extraction, and test surface drying; (iii) uses vacuum to direct fluid to flow through or over or around the optically active surface and into a waste adsorbent material or reservoir within the cartridge; and (iv) incubates a sample, or a component thereof, on the surface of the optically active surface for a period of time under normal gravity conditions prior to reagent transfer through or around the optically active surface by vacuum.
Preferably, the vacuum source maintains a weak (about 20 mm Hg to about 40 mm Hg; preferably about 30 mm Hg) vacuum when the test surface is positioned for reading through the optical reading well. In other preferred embodiments, the vacuum source is disengaged during incubations on the test surface and/or when the reagent carousel is indexed. The vacuum level can preferably be raised to between about 120 mm Hg and about 180 mm Hg, most preferably about 150 mm Hg, in order to dry the test surface prior to reading, to draw extraction reagent from a swab, or to draw a fluid through a concentrating membrane prior to extraction. One skilled in the art will recognize that the vacuum required for reagent flow can vary, depending on the composition of the reagent and the composition and porosity of a membrane, filter, or test surface. A feedback loop to vary the vacuum level dependent upon sample or test conditions may be incorporated to automatically adjust the vacuum in the device to accommodate a number of parameters affecting reagent flow.
In further embodiments, a series of pneumatic valves may be added under the cartridge receiving stage of the instrument. These pneumatic valves allow the introduction of air into or over various parts of the cartridge or instrument. The air flow may be directed onto the optically active test surface to assist the vacuum system in drying the test surface. Or the air flow may be used to assist in fluid movement.
In other particularly preferred embodiments: (i) the instrument uses fixed angle ellipsometry as the optical analysis feature; (ii) the instrument comprises an LED light source; (iii) the LED light source emits light at 525 nm; (iv) the LED light source is positioned at a 20xc2x0 angle of incidence relative to a line normal to the plane of the optically active test surface; (v) a photodiode detector is positioned at a 20xc2x0 angle of detection relative to a line normal to the plane of the optically active test surface; (vi) polarizing and analyzing polarizers are positioned at 90xc2x0 relative to one another; and (vii) the instrument allows for synchronous detection to eliminate stray light as a source of noise.
The term xe2x80x9clight sourcexe2x80x9d as used herein refers to any source of electromagnetic radiation. Electromagnetic radiation can also be referred to as xe2x80x9clight.xe2x80x9d Such electromagnetic radiation may include wavelengths from about 10xe2x88x926 xcexcm to about 108 xcexcm; preferred is electromagnetic radiation from the ultraviolet to infrared wavelengths; particularly preferred electromagnetic radiation is visible light. Suitable light sources are well known to those skilled in the art, and can include any source of monochromatic or polychromatic radiation. The use of monochromatic radiation is preferred. The terms xe2x80x9cmonochromatic radiationxe2x80x9d or xe2x80x9cmonochromaticxe2x80x9d light as used herein refer to electromagnetic radiation having a bandwidth that is sufficiently narrow to function as a single wavelength for design purposes. Preferred light sources are lasers, laser diodes, and light emitting diodes (LEDs). In preferred embodiments, an aperture, preferably a bar-shaped aperture, is placed in the optical path, oriented parallel to a stripe-shaped capture zone on the test surface. The aperture can provide a larger interrogation area of the test surface, rendering the detector less susceptible to surface variations in the test surface and providing a larger area over which signal is averaged.
As used herein, the term xe2x80x9cdetectorxe2x80x9d refers to any device for detecting electromagnetic radiation by the production of electrical or optical signals, and includes color sensors, color detectors, image detectors, spectrophotometers, luminometers, fluorometers, potentiometers, interferometers, polarimeters, and ellipsometers, whether these detectors are driven to provide analog or digital signals, as well as any other light detection device. Preferred detectors detect electromagnetic radiation, particularly visible light, with the resulting production of electrical or optical signals. A signal processing element can process these signals to yield this information, for example by the use of standard curves, to associate the signals with an optical film thickness. In especially preferred embodiments, the optical film thickness is interpreted as a binding assay result, e.g., the result of a test showing either a positive, negative, or inconclusive result in a test for a specific analyte.
Preferably, the light source and detector of the instrument are positioned at an angle of incidence of between about 10xc2x0 and about 40xc2x0 relative to a line normal to the plane of the optically active test surface. Most preferably, the angle of incidence is about 10xc2x0, 20xc2x0, 30xc2x0, and 40xc2x0.
The term xe2x80x9cpolarizerxe2x80x9d as used herein refers to a device that receives incoming electromagnetic radiation, and produces therefrom radiation which is polarized. Suitable polarizers, such as polarizing filters and analyzers, are well known to those skilled in the art. As described herein, polarizers can be positioned to polarize incoming light from the light source prior to contact with the sample under study, as well as light reflected from the sample under study. A polarizer can be fixed within an optical pathway. Alternatively, one or more of the polarizers can include a mechanism for varying s- and p- components of polarized light with time by rotating the polarization element, or a component thereof, on its optical axis. Preferably, this mechanism rotates a polarizing filter that is located in the position of a polarizer or analyzer in a conventional ellipsometer. Rotation of a polarizing filter provides a corresponding quasi-sinusoidal intensity in the electromagnetic radiation that is reflected from the sample under study.
The terms xe2x80x9cpolarizing polarizerxe2x80x9d and xe2x80x9canalyzing polarizerxe2x80x9d as used herein refer to polarizers which interact with incident light prior to and following light impinging on the optically active test surface. In preferred embodiments, the polarizing and analyzing polarizers are set at about 70xc2x0 to about 110xc2x0 relative to one another. Most preferably, the polarizing and analyzing polarizers are set at about 70xc2x0, 80xc2x0, 90xc2x0, 100xc2x0, and 110xc2x0 relative to one another.
The term xe2x80x9clinear polarizationxe2x80x9d as used herein refers to a polarization state that is essentially all s-polarization or all p-polarization. Electromagnetic radiation is linearly polarized if, in either linear state, there is not enough of the other polarization state to affect the outcome of the measurement. Preferably, a linear polarizing filter may be rotated up to about 20xc2x0 rotation off of its optical axis without introducing appreciable measurement errors.
In another aspect, the invention concerns methods of determining the presence or amount of an analyte in a sample. The method comprises providing an assay cartridge and an analytical instrument as defined herein, placing the sample into the sample receiving port of the assay cartridge, placing the assay cartridge into the receiving mechanism of the analytical instrument, performing an assay using the analytical instrument according to an assay algorithm, and using the signal processor to determine the presence or amount of the analyte in the sample.
In particularly preferred embodiments, a sample is selected from the group consisting of a throat swab, a vaginal swab, an endocervical swab, a rectal swab, a urethral swab, a nasal swab, a nasopharyngeal swab, a fluid, water, urine, blood, sputum, serum, plasma, an aspirate, a wash, a tissue homogenate, and a process fluid.
The preferred methods of using the instrument and cartridge include methods for analyzing the data and reporting a result. Preferred methods of use will be described in terms of a reagent or assay cartridge that is designed to detect an analyte on an optically active test surface where the optical detection system is based on a fixed polarizer ellipsometer. Those skilled in the art will recognize that methods for use of the instrument and the reagent cartridge will be similar for any other combination of analyte, test surface, and detection system.
In using the system, a user selects the reagent cartridge designed for the analyte of interest, for example a reagent cartridge designed to detect a particular microorganism. In particularly preferred embodiments, a microorganism is a bacterium, a virus, or a fungus, and most preferably a pathogenic bacterium, virus, or fungus. The user provides a specimen from the patient to be tested for the analyte of interest, collected, for example, using a throat swab. The user enters or scans (e.g., by bar code) the specimen identification number and the reagent cartridge lot information. Alternatively, the instrument may read the bar code on the top, side, or bottom of the cartridge when it is placed in the instrument. A bar code, or other identifying element of the cartridge, can provide information to a xe2x80x9cDallasxe2x80x9d chip which provides the instrument with assay a quality control parameters, a lot number, a sample type, etc. The user places the throat swab in the sample receiving port in the reagent cartridge and loads the cartridge into the instrument. The instrument may close a door behind the reagent cartridge for orientation and stability or it may pull the cartridge into the appropriate slot with a cassette player type mechanism. The cartridge may be placed or set on alignment or set pins formed on and above the cartridge receiving stage. The set pins assure that the cartridge is properly oreinted. The rotation element may serve as one of the set pins. Preferably, the instrument receives the cartridge in proper registration and alignment so that the cartridge may be mechanically indexed through the appropriate sequence of reagent additions and incubation steps. The user may then enter a xe2x80x9cstart analysisxe2x80x9d command via the user interface of the instrument, or the optical sensor that detects the presence of a cartridge may cause the initiation of the assay protocol.
The instrument then rotates the reagent carousel into a position so that the optically active surface may be scanned to provide a baseline reading. The optical scanning procedure may be conducted on one or more fixed points within the optical reading well or may be a linear segment of the optically active test surface or may analyze the complete test surface. The optical detection system does not move but the cartridge may be linearly displaced to expose a new section of the optically active test surface at each reading point.
A possible reading scheme occurs as follows. The collected data is stored for use in the final data analysis routine. For the baseline scan the cartridge is rotated to the optical read window. The cartridge is moved along the instruments positive y-axis so that the beam spot is positioned on the outer edge of the read area""s periphery. The cartridge is moved along the negative y-axis and readings are taken every 12.7 microns. The sample size is dependent on the beam size and configuration and the number of samples per unit area needed to provide the accuracy desired in the final result. The raw data is stored in a file. The first column of raw data is the position of the read area on the optically active test surface. The second column is the corresponding reflected signal in millivolts for the baseline scan. The same process is repeated at appropriate assay steps. The multiple reads provide QC checks of the test cartridge and can stop a test if the readings at a specific stage fall out of a pre-set range. The multiple reads per assay allow for a test generating high noise to be rejected earlier in the analysis process.
The data analysis software can then align multiple scans of the same surface by selecting an edge feature to align each scan relative to the other scans, and thus provide for proper data comparisons. The edge features can be eliminated from the data analysis routine. The readings can be taken at any index distance desired and the degree of overlap selected to provide the most accurate level of result. It should, however, be set at the minimal acceptable value as the number of measurements made will also affect the time to result. The final method of data analysis can be tailored to the type of test method used, the required accuracy and precision, and other parameters determined by the intended use of the test result. Acceptable data analysis routines are known to those skilled in the art but could include peak to peak comparisons, peak smoothing, or other methods for normalizing the collected data or methods for data reduction. Another option might be to do image processing of the surface. In this case, each scan taken will visualize the entire test surface. Images would be compared between scans and appropriate data selected to provide the final test result. One or more of the detection scans described in the previous procedure may not be required for all detection methods and detection surfaces. However, one or more scan is required to provide sufficient information for data normalization.
Once the baseline scan is complete, the instrument activates the extraction reagent well and causes an extraction reagent to flow into the sample receiving port. The reagent carousel will be indexed by rotation to align the sample receiving port over the optically active test surface. Following a pre-set extraction period, the vacuum system can draw the sample fluid from the sample collection device through a filter to remove particulates, and onto the optically active test surface. Vacuum must be applied to the sample processing element such that sample fluid or processed sample fluid is drawn through the filter of the sample-processing element and onto the optically active test surface without being drawn through the optically active test surface. This is achieved by placement of a vacuum port between the sample-processing element and the optically active test surface so that the fluid will only flow to the optically active test surface and not through, over, or around it.
Following sample addition and a pre-set incubation, the reagent carousel rotates to align the proper reagent well with the optically active test surface. The sample may be added in the presence of a neutralizing agent that is added to the test surface prior to the addition of sample from an appropriate reagent well. The plunger mechanism of the instrument forces the piston to break the reagent seal and deliver the fluid to the optically active test surface. By using the piston and plunger mechanism, the flow rate of reagent delivered to the test surface can be controlled. Fluid is positively displaced by the piston and delivered to the optically active test surface under gravity and positive displacement. In this case positive displacement does not include any aspiration or introduction of air but is a mechanical method for positive displacement. The reagent will combine with the test sample on the surface of the optically active test surface. Following a pre-set, static incubation period, the fluid will be drawn through or over or around the optically active test surface by activation of the vacuum system and all liquid waste is retained within the assay cartridge. When analyte is present in the neutralized sample, analyte will bind in one or more positions to the analyte-specific binding layer on the optically active test surface. After specific reagent additions, the test surface may be washed with a solution from one or more reagent wells to remove any unreacted reagents. After wash steps the test surface may be dried by a combination of vacuum and air flow, by vacuum alone, or by air flow alone.
Next, a new reagent well aligns over the optically active test surface and a wash solution is delivered. All reagent additions occur with the vacuum system in the disengaged position. Once the reagent is delivered and the pre-set period is past, the vacuum system is engaged to draw the fluid through or around or over the optically active test surface. Reagent removal is controlled by application of the vacuum. The static incubation improves assay performance while the flow through process simplifies assay processing. The test surface may be rinsed with one or more wash reagents from one or more reagent well. Once the optically active test surface is rinsed, the reagent carousel is rotated to the optical reading well and the optically active test surface is scanned again to look for non-specific binding and debris from the sample and to qualify the optically active test surface integrity. A scan following the addition of sample is required to assist in normalizing the data. An amplifying or signal-generating may also be added to the test surface together with the analyte when appropriate for the type of test surface employed.
Upon completion of the second optical scan, an amplifying reagent, or signal generating reagent (depending on the test surface) can be applied to the optically active test surface by rotation of the appropriate reagent well over the test surface. The amplifying reagent is allowed to react for a period of time and then the vacuum system is engaged to draw the unreacted reagent through or around or over the optically active test surface. The reagent carousel is again rotated to align a reagent well over the test surface and a wash solution applied. The reagent seal is pierced as with the original reagent delivery and the vacuum is engaged at the appropriate time. The vacuum will serve to dry the optically active test surface. A small air-flow device may be included to improve the speed of the surface drying. When a test surface is not optically active then this final drying step may not be required.
Once the optically active test surface is washed and dried then a final optical scan is conducted. The optical reading well of the reagent carousel is rotated over the test surface and the scan conducted. The optical scans may occur with the vacuum system engaged if no distortion of the test surface occurs under vacuum. Positive report of analyte binding is provided only when sufficient signal intensity is observed and the proper sequence of elements on the test surface are identified.
The instrument component list preferably includes an optical detection element preferably contained in a single common unit that may be attached to the instrument support structure; a plunger assembly that again is a single unit with all of the required functionalities built in; a cartridge carriage unit that provides for orientation, retention, and positioning of the cartridge within the final assembled instrument; a support structure designed to position and stabilize all of the functional units of the instrument and which may assist in placement and movement functions of the instrument; a vacuum system; one or more motors to drive the positioning of the plunger and the cartridge, etc; and electronic components to control, monitor, and report on the various functions and measurements made by the instrument.
Most preferably, the assay cartridge can be manufactured as follows. The cartridge consists of the following molded pieces: the carousel, the pistons (one or more designs), the test surface holder (top), the waste reservoir holder (bottom), the sample receiving port, and an attachable hinged swab retention element (when required). The test surface holder has an opening and the optically active test surface is heat sealed to the bottom of the opening so that the optical surface is exposed through the opening. The optically active test surface is created by applying the optical coatings and other layers required for proper optical function and then coated with the appropriate analyte specific capture reagents before it is ready to attach to the test surface holder. The waste reservoir has positions for one or more adsorbent pads of highly adsorbent material to be placed within the wells in the floor of the part. These two pieces may be heat sealed, glued, or snapped together to provide the platform piece of the final assembled cartridge. The reagent carousel has the upper vapor seal applied by heat sealing the polyethylene layer of the vapor seal to the plastic carousel at a number of points, e.g., around each reagent well and at the edges of the carousel. The plastic pistons are loaded onto the reagent well and then the reagent well is filled with reagent. Then the lower vapor seal is then applied to the cartridge. The lower vapor seal has an opening that corresponds to the sample receiving port and a cutout that corresponds to the reading well. At the under side of the carousel and where the vapor seal has an opening one or more membranes (gradient membranes, single-pore size membranes, Memtex(copyright) membranes, etc.) are heat sealed to the under surface of the sample receiving port and then an adhesive, plastic gasket is applied over the membranes to assist in the establishing of vacuum during use. If the sample receiving feature is not an integral part of the mold then the element can feature a snap fit into position within the carousel. The carousel is then attached to the lower platform element of the cartridge. The entire cartridge may be wrapped in a vapor proof bag or may be placed in a kit as individual unwrapped units.